Figure 3. Mechanism of sorafenib-mediated radioprotection in vitro.

HepG2 cells were synchronized then re-fed with complete medium (10% serum) either containing 5 µM sorafenib (SOR) or vehicle control (DMSO). (A) Percent of cells in G₁, S, and G₂ phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G₁-S delay and cell cycle slowing in synchronized HepG2 cells, causing more cells to be in G₁-S versus G₂-M when radiation would be delivered 24 h after beginning incubation with SOR. (B & C) Unsynchronized Hep3b and HCC-4-4 cells were exposed to SOR or vehicle control for 24 h and then fixed with ethanol for cell cycle analysis. Percent of cells in G₁, S, and G₂ phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G₁-S delay in both cell lines and reduced the number of cells in G₂-M when radiation would be delivered at 24 h after beginning incubation with SOR. Asterisks denote significant differences between corresponding columns in the control and SOR arms for each cell line by Student's t-test. Data for the HuH7 cell line is not shown because it was found to exhibit polyploidy; these data are displayed in Figure S2. Data for unsynchronized HepG2 cells are also shown in Figure S2. All experiments were done in triplicate and repeated. (D) Immunoblotting for phospho-p53 and p21 after treatment of HepG2 cells with each of the 5 different treatment arms (control—incubation with DMSO for 12 hours; SOR—incubation with 5-µM sorafenib for 12 hours; RT—incubation with DMSO for 12 hours with irradiation at 6-hour midpoint; CONC—incubation with 5-µM sorafenib for 12 hours with irradiation at 6-hour midpoint; SEQ—incubation with DMSO for 6 hours, irradiation, followed by incubation with 5-µM sorafenib for 6 hours). All irradiation doses were single fractions of 6 Gy. Corresponding immunoblot data for the remaining 3 cell lines can be found in Figure S2C.